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1.
Mol Cell Endocrinol ; 581: 112107, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-37981188

RESUMO

Compartmentalization of GPCR signaling is an emerging topic that highlights the physiological relevance of spatial bias in signaling. The parathyroid hormone (PTH) type 1 receptor (PTH1R) was the first GPCR described to signal via heterotrimeric G-protein and cAMP from endosomes after ß-arrestin mediated internalization, challenging the canonical GPCR signaling model which established that signaling is terminated by receptor internalization. More than a decade later, many other GPCRs have been shown to signal from endosomes via cAMP, and recent studies have proposed that location of cAMP generation impacts physiological outcomes of GPCR signaling. Here, we review the extensive literature regarding PTH1R endosomal signaling via cAMP, the mechanisms that regulate endosomal generation of cAMP, and the implications of spatial bias in PTH1R physiological functions.


Assuntos
Arrestinas , Receptor Tipo 1 de Hormônio Paratireóideo , Arrestinas/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Transdução de Sinais/fisiologia , Hormônio Paratireóideo/metabolismo , Biologia
2.
Am J Physiol Cell Physiol ; 323(3): C783-C790, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35912987

RESUMO

The canonical model for G protein-coupled receptors (GPCRs) activation assumes that stimulation of heterotrimeric G protein signaling upon ligand binding occurs solely at the cell surface and that duration of the stimulation is transient to prevent overstimulation. In this model, GPCR signaling is turned-off by receptor phosphorylation via GPCR kinases (GRKs) and subsequent recruitment of ß-arrestins, resulting in receptor internalization into endosomes. Internalized receptors can then recycle back to the cell surface or be trafficked to lysosomes for degradation. However, over the last decade, this model has been extended by discovering that some internalized GPCRs continue to signal via G proteins from endosomes. This is the case for the parathyroid hormone (PTH) type 1 receptor (PTHR), which engages on sustained cAMP signaling from endosomes upon PTH stimulation. Accumulative evidence shows that the location of signaling has an impact on the physiological effects of GPCR signaling. This mini-review discusses recent insights into the mechanisms of PTHR endosomal signaling and its physiological impact.


Assuntos
Arrestinas , Receptor Tipo 1 de Hormônio Paratireóideo , Arrestinas/metabolismo , AMP Cíclico/metabolismo , Hormônio Paratireóideo/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismo
3.
J Biol Chem ; 298(9): 102332, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35933010

RESUMO

The parathyroid hormone (PTH)-related protein (PTHrP) is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling, and causes hypercalcemia in patients with malignancy. Although mature forms of PTHrP in the body consist of splice variants of 139, 141, and 173 amino acids, our current understanding on how endogenous PTHrP transduces signals through its cognate G-protein coupled receptor (GPCR), the PTH type 1 receptor (PTHR), is largely derived from studies done with its N-terminal fragment, PTHrP1-36. Here, we demonstrate using various fluorescence imaging approaches at the single cell level to measure kinetics of (i) receptor activation, (ii) receptor signaling via Gs and Gq, and (iii) receptor internalization and recycling that the native PTHrP1-141 displays biased agonist signaling properties that are not mimicked by PTHrP1-36. Although PTHrP1-36 induces transient cAMP production, acute intracellular Ca2+ (iCa2+) release and ß-arrestin recruitment mediated by ligand-PTHR interactions at the plasma membrane, PTHrP1-141 triggers sustained cAMP signaling from the plasma membrane and fails to stimulate iCa2+ release and recruit ß-arrestin. Furthermore, we show that the molecular basis for biased signaling differences between PTHrP1-36 and properties of native PTHrP1-141 are caused by the stabilization of a singular PTHR conformation and PTHrP1-141 sensitivity to heparin, a sulfated glycosaminoglycan. Taken together, our results contribute to a better understanding of the biased signaling process of a native protein hormone acting in conjunction with a GPCR.


Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo , AMP Cíclico/metabolismo , Heparina/metabolismo , Humanos , Ligantes , Conformação Proteica , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo
4.
Nat Chem Biol ; 18(3): 272-280, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34949836

RESUMO

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.


Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo , Receptores Acoplados a Proteínas G , Ligantes , Simulação de Dinâmica Molecular , Transdução de Sinais
5.
Sci Signal ; 14(703): eabc5944, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34609896

RESUMO

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein­coupled receptor (GPCR) that regulates mineral ion, vitamin D, and bone homeostasis. Activation of the PTHR by PTH induces both transient cell surface and sustained endosomal cAMP production. To address whether the spatial (location) or temporal (duration) dimension of PTHR-induced cAMP encodes distinct biological outcomes, we engineered a biased PTHR ligand (PTH7d) that elicits cAMP production at the plasma membrane but not at endosomes. PTH7d stabilized a unique active PTHR conformation that mediated sustained cAMP signaling at the plasma membrane due to impaired ß-arrestin coupling to the receptor. Experiments in cells and mice revealed that sustained cAMP production by cell surface PTHR failed to mimic the pharmacological effects of sustained endosomal cAMP production on the abundance of the rate-limiting hydroxylase catalyzing the formation of active vitamin D, as well as increases in circulating active vitamin D and Ca2+ and in bone formation in mice. Thus, similar amounts of cAMP generated by PTHR for similar lengths of time in different cellular locations, plasma membrane and endosomes, mediate distinct physiological responses. These results unveil subcellular signaling location as a means to achieve specificity in PTHR-mediated biological outcomes and raise the prospect of rational drug design based upon spatiotemporal manipulation of GPCR signaling.


Assuntos
Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos , AMP Cíclico
6.
J Biol Chem ; 297(3): 101118, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34437901

RESUMO

cAMP is the indispensable second messenger regulating cell metabolism and function in response to extracellular hormones and neurotransmitters. cAMP is produced via the activation of G protein-coupled receptors located at both the cell surface and inside the cell. Recently, Tsvetanova et al. explored cAMP generation in distinct locations and the impact on respective cell functions. Using a phospho-proteomic analysis, they provide insight into the unique role of localized cAMP production in cellular phospho-responses.


Assuntos
AMP Cíclico , Proteômica , Receptores Acoplados a Proteínas G , Sistemas do Segundo Mensageiro , Transdução de Sinais
7.
Proc Natl Acad Sci U S A ; 117(13): 7455-7460, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32184323

RESUMO

cAMP production upon activation of Gs by G protein-coupled receptors has classically been considered to be plasma membrane-delimited, but a shift in this paradigm has occurred in recent years with the identification of several receptors that continue to signal from early endosomes after internalization. The molecular mechanisms regulating this aspect of signaling remain incompletely understood. Here, we investigated the role of Gq/11 activation by the parathyroid hormone (PTH) type 1 receptor (PTHR) in mediating endosomal cAMP responses. Inhibition of Gq/11 signaling by FR900359 markedly reduced the duration of PTH-induced cAMP production, and this effect was mimicked in cells lacking endogenous Gαq/11 We determined that modulation of cAMP generation by Gq/11 occurs at the level of the heterotrimeric G protein via liberation of cell surface Gßγ subunits, which, in turn, act in a phosphoinositide-3 kinase-dependent manner to promote the assembly of PTHR-ßarrestin-Gßγ signaling complexes that mediate endosomal cAMP responses. These results unveil insights into the spatiotemporal regulation of Gs-dependent cAMP signaling.


Assuntos
AMP Cíclico/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Animais , Arrestinas/metabolismo , Membrana Celular/metabolismo , Depsipeptídeos/farmacologia , Endossomos/metabolismo , Células HEK293 , Humanos , Camundongos , Osteoblastos/metabolismo , Hormônio Paratireóideo/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Cultura Primária de Células , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismo
8.
Curr Protoc Cytom ; 83: 12.2.1-12.2.14, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29345327

RESUMO

Modern digital microscopy combines the equipment of classical light microscopy with a computerized imaging system. The technique comprises image formation by optics, image registration by a camera, and saving of image data in a computer file. This chapter describes limitations that are particular to each of these processes, including optical resolution, efficiency of image registration, characteristics of image file formats, and data management. Further suggestions are given which serve, in turn, to help construct a set of guidelines aimed at optimization of digital microscopic imaging. © 2018 by John Wiley & Sons, Inc.


Assuntos
Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Microscopia/instrumentação , Microscopia/métodos
9.
Cell Calcium ; 60(5): 356-362, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27593159

RESUMO

Oxidative stress drives cell death in a number of diseases including ischemic stroke and neurodegenerative diseases. A better understanding of how cells recover from oxidative stress is likely to lead to better treatments for stroke and other diseases. The recent evidence obtained in several models ties the process of lysosomal exocytosis to the clearance of protein aggregates and toxic metals. The mechanisms that regulate lysosomal exocytosis, under normal or pathological conditions, are only beginning to emerge. Here we provide evidence for the biphasic effect of oxidative stress on lysosomal exocytosis. Lysosomal exocytosis was measured using the extracellular levels of the lysosomal enzyme beta-hexosaminidase (ß-hex). Low levels or oxidative stress stimulated lysosomal exocytosis, but inhibited it at high levels. Deletion of the lysosomal ion channel TRPML1 eliminated the stimulatory effect of low levels of oxidative stress. The inhibitory effects of oxidative stress appear to target the component of lysosomal exocytosis that is driven by extracellular Ca2+. We propose that while moderate oxidative stress promotes cellular repair by stimulating lysosomal exocytosis, at high levels oxidative stress has a dual pathological effect: it directly causes cell damage and impairs damage repair by inhibiting lysosomal exocytosis. Harnessing these adaptive mechanisms may point to pharmacological interventions for diseases involving oxidative proteotoxicity or metal toxicity.


Assuntos
Exocitose , Lisossomos/metabolismo , Estresse Oxidativo , Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Células HeLa , Humanos , Lisossomos/efeitos dos fármacos , Metais Pesados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/deficiência , Canais de Potencial de Receptor Transitório/metabolismo , Células Tumorais Cultivadas
10.
Dis Model Mech ; 8(12): 1591-601, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26398942

RESUMO

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the MCOLN1 gene, which encodes the lysosomal transient receptor potential ion channel mucolipin-1 (TRPML1). MLIV causes impaired motor and cognitive development, progressive loss of vision and gastric achlorhydria. How loss of TRPML1 leads to severe psychomotor retardation is currently unknown, and there is no therapy for MLIV. White matter abnormalities and a hypoplastic corpus callosum are the major hallmarks of MLIV brain pathology. Here, we report that loss of TRPML1 in mice results in developmental aberrations of brain myelination as a result of deficient maturation and loss of oligodendrocytes. Defective myelination is evident in Mcoln1(-/-) mice at postnatal day 10, an active stage of postnatal myelination in the mouse brain. Expression of mature oligodendrocyte markers is reduced in Mcoln1(-/-) mice at postnatal day 10 and remains lower throughout the course of the disease. We observed reduced Perls' staining in Mcoln1(-/-) brain, indicating lower levels of ferric iron. Total iron content in unperfused brain is not significantly different between Mcoln1(-/-) and wild-type littermate mice, suggesting that the observed maturation delay or loss of oligodendrocytes might be caused by impaired iron handling, rather than by global iron deficiency. Overall, these data emphasize a developmental rather than a degenerative disease course in MLIV, and suggest that there should be a stronger focus on oligodendrocyte maturation and survival to better understand MLIV pathogenesis and aid treatment development.


Assuntos
Encéfalo/metabolismo , Ferro/metabolismo , Mucolipidoses/metabolismo , Mucolipidoses/patologia , Bainha de Mielina/patologia , Animais , Axônios/patologia , Encéfalo/patologia , Contagem de Células , Corpo Caloso/patologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/metabolismo , Oligodendroglia/patologia , Estresse Oxidativo , Canais de Potencial de Receptor Transitório/deficiência , Canais de Potencial de Receptor Transitório/genética
11.
Biochem J ; 470(1): 65-76, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26251447

RESUMO

Transition metal toxicity is an important factor in the pathogenesis of numerous human disorders, including neurodegenerative diseases. Lysosomes have emerged as important factors in transition metal toxicity because they handle transition metals via endocytosis, autophagy, absorption from the cytoplasm and exocytosis. Transcription factor EB (TFEB) regulates lysosomal biogenesis and the expression of lysosomal proteins in response to lysosomal and/or metabolic stresses. Since transition metals cause lysosomal dysfunction, we proposed that TFEB may be activated to drive gene expression in response to transition metal exposure and that such activation may influence transition metal toxicity. We found that transition metals copper (Cu) and iron (Fe) activate recombinant TFEB and stimulate the expression of TFEB-dependent genes in TFEB-overexpressing cells. In cells that show robust lysosomal exocytosis, TFEB was cytoprotective at moderate levels of Cu exposure, decreasing oxidative stress as reported by the expression of heme oxygenase-1 (HMOX1) gene. However, at high levels of Cu exposure, particularly in cells with low levels of lysosomal exocytosis, activation of overexpressed TFEB was toxic, increasing oxidative stress and mitochondrial damage. Based on these data, we conclude that TFEB-driven gene network is a component of the cellular response to transition metals. These data suggest limitations and disadvantages of TFEB overexpression as a therapeutic approach.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/biossíntese , Cloretos/farmacologia , Cobre/farmacologia , Compostos Férricos/farmacologia , Elementos de Transição/farmacologia , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia
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